Mammalian cells take up, and release physiological nucleosides and many of their synthetic analogs primarily by means of specific integral plasma membrane glycoproteins known as nucleoside transporters (Paterson and Cass, 1986; Plagemann et al., 1988; Gati and Paterson, 1989; Paterson et al., 1991; Cass, 1995; Thorn and Jarvis, 1996). Nucleoside transporters have been classified into two categories: (i) equilibrative (facilitated diffusion) and (ii) concentrative (secondary active) sodium-dependent. Two equilibrative transporters with similar broad substrate specificities have been identified and designated as the es (equilibrative sensitive) and ei (equilibrative insensitive) transporters, on the basis of their sensitivity or insensitivity to inhibition by nitrobenzylthioinosine (NBMPR, 1), respectively. Six sodium ion-coupled (concentrative) transporters designated cif/N1, cit/N2, cib/N3, cit/N4, cs/N5 and csg/N6 have also been identified in mammalian tissues (Cass, 1995, Young et al., 2000). However, sodium-dependent nucleoside transport is only a minor component of mammalian tissues and plays a role mainly in secretory tissues. The es transporter is by far the major nucleoside transporter of most mammalian tissues, especially heart tissue (Williams, 1996; Hoehner et al., 1996; Abd-Elfattah et al., 1998a), and is highly sensitive to inhibition by NBMPR, and related purine 6-position (nitrobenzyl) nucleosides (Paul et al., 1975; Robins et al., 1994; Paterson et al., 1983), and has a high affinity for NBMPR (Kd in the 0.1-1.0 nM range).

In addition to their role as precursors for salvage synthesis of nucleotides used for DNA and RNA synthesis, physiological nucleosides are also involved in signal transduction and metabolic pathways. Adenosine is especially involved in protecting tissues from ischemic and inflammatory damage (Engler, 1987; Ohta and Sitkovsky, 2001). Adenosine's tissue protective effects can be harnessed for the treatment of ischemic heart disease and stroke, as wells as other ischemic conditions and the preservation of donor hearts and kidneys (reviewed in Buolamwini, 1997). Agents that potentiate endogenous adenosine's protective effects are therefore being pursued. One attractive potential strategy for in vivo adenosine potentiation is adenosine transport blockade (Van Belle, 1993a, 1993b). Adenosine is automatically released endogenously in myocardial infarction or stroke, as a “retaliatory” metabolite (Newby, 1984) through ATP catabolism to protect against ischemic tissue damage through its interaction with cell surface G-protein coupled adenosine receptors (Ver Donck, 1994; Linden, 2001). However, adenosine's protective effects are quickly lost mainly by cellular uptake through nucleoside transporters (Van Belle, 1993a), which contribute to its depletion from the extracellular milieu. The adenosine potentiation effects of nucleoside transporter inhibitors, which help maintain extracellular adenosine concentrations, have been demonstrated in heart and brain ischemia models (Van Belle, 1993a, 1993b; Abd Elfattah and Wechsler, 1994; Abd-Elfattah et al., 1998b; Rudolphi et al., 1992; Parlinson et al., 2000; Zhang et al., 2002). The benefit of using NT inhibitors rather than adenosine receptor agonists is that the effects of NT inhibitors, unlike adenosine receptor agonists are localized (Van Belle, 1993b) to tissues where adenosine is released locally in an ischemic episode, making this approach event- and site-specific. Adenosine receptor agonists on the other hand will trigger adenosine receptors all over the body leading to unwanted side effects, a major limitation in the drug development of adenosine receptor agonists (Erion, 1993). Other less attractive adenosine enhancement strategies include the use of adenosine deaminase (ADA) inhibitors such as deoxycoformycin (Phillis and O'Regan, 1996). The disadvantage in using ADA inhibitors is that they are prone to cause severe combined immunodeficiency (SCID), a condition that is observed in a genetic deficiency in the production of the enzyme (Strachan and Read, 1996).
The combination of NT inhibitors with de novo synthesis inhibitor antimetabolite chemotherapy in cancer and infectious diseases is also of considerable interest (Buolamwini, 1997). Thus, dipyridamole has been shown to enhance the antitumor effects of methotrexate (Cabral et al., 1984) and 5-fluorouracil (Grem and Fischer, 1985). The experimental and clinical studies relating to the modulation of 5-fluorouracil by nucleoside transport inhibitors have been reviewed (Tew et al., 1993). The continued interest in this therapeutic approach is shown by a recent prospective randomized clinical trial that tested the efficacy of a combination of orally administered dipyridamole with leucovorin and 5-FU in advanced colorectal cancer (Kohne et al., 1995).
The available NT inhibitors, however, lack the requisite pharmacological profiles are toxic, mutagenic, ineffective in vivo, or nonspecific (reviewed in Buolamwini, 1997), and therefore the above-mentioned therapeutic strategies will benefit tremendously from the discovery of better nucleoside transport inhibitors.
In this regard, the compounds that are the subject of this disclosure are invented to accomplish two goals: (i) to provide conformationally restrained analogs of NBMPR to be used as probes of the bioactive conformation of NBMPR for better rational nucleoside transporter inhibitor design and (ii) to overcome a major disadvantage of NBMPR, which has to do with the lability of the S6-nitrobenzyl substituent, which is easily lost in vivo to drastically reduce NT inhibitory activity (up to a 1000 fold). This lability has actually allowed NBMPR to be used as an effective photolabeling reagent for the es nucleoside transporter (Young et al., 1983; Boumah et al., 1992). Incorporating the high potency conferring nitrobenzyl group of NBMPR into a nitrotetrahydroisoquinoline system prevents it from being easily cleaved. At the same time this molecular modification restrains the conformational flexibility at the purine 6-position substituents and helps to better map the orientation of the nitrobenzyl group when bound to the es transporter.
Novel compounds are synthesized and their binding affinity at the es nucleoside transporter evaluated by a flow cytometric assay (Buolamwini et al., 1994). Their inhibition of nucleoside uptake has also been demonstrated, as well as their ability to enhance the recovery of isolated heart preparation from global ischemia.